四株深海来源的稀有放线菌抗肿瘤活性代谢产物的研究

Marine microorganisms have been considered as important source of anti-tumor lead compounds. In our effort to search for antitumor secondary metabolites from marine-derived actinomycetes, we found that the fermentation extract from three strains of Micromonospora and a strain of Rhodococcus exhibited cytotoxic activity. In this research proposal, we are planning to investigate the secondary metabolites of these rare actinomycetes for the purpose of discovering anti-tumor lead compounds. A series of methods will be applied, including optimization of culture medium and fermentation conditions, extract and purification, structure identification, in vitro and in vivo bioactive assays. First, the culture medium ingredients and fermentation conditions will be optimized. Then bench-scale fermentation will be carried out. The fermentation product will be extracted with organic solvent, and the extract will be isolated by bioassay guided fractionation using MPLC and HPLC chromotography equipped with silica gel or ODS column. The ESI-MS and NMR data for purified compounds will be detected and compared with those in MarinLit database to identify known compounds quickly. Structures of new compounds will be characterized via comprehensive analyses of spectroscopica data, including MS, 1D and 2D NMR. The stereochemistry will be determined on the basis of single crystal x-ray diffraction technique, and Mosher's and Marfey's methods. The cytotoxic activity of the purified compounds will be evaluated using SRB or MTT assays against a panel of human tumor cell lines. These compounds with significant cytotoxicity will be examined for their in vivo antitumor effect using xenograft nude mouse model. The results of this project will develop new molecular entity for anticancer therapeutics, and provide a new way for efficient utilization of marine microorganism resources.

海洋微生物已经成为抗肿瘤活性天然产物的重要源泉。在前期的筛选中，我们发现深海来源的三株小单孢及一株红球菌的发酵产物具有很强的细胞毒活性，本项目以这四株稀有放线菌为研究对象，采用"发酵优化-发酵培养-分离纯化-结构鉴定-体外筛选-体内评价"的技术路线，发掘抗肿瘤先导化合物。首先优化四株稀有放线菌发酵培养基组成和发酵条件，继而进行实验室规模发酵，在活性跟踪条件下以中压柱层析和制备HPLC快速分离次级代谢产物；利用ESI-MS/NMR 与数据库对比进行快速排重，综合分析各种波谱数据鉴定新化合物的结构,通过NOESY谱、X-单晶衍射、化学衍生等确定其立体构型；对分离获得的单体化合物采用SRB或者MTT法进行细胞毒活性筛选，利用荷瘤裸鼠模型对活性显著的化合物进行抗肿瘤药效评价，发现抗肿瘤先导化合物。本项目的实施，将为抗肿瘤药物研发提供物质基础，为海洋微生物资源的高效利用开辟新途径。

本项目以南海海洋来源的放线菌为研究对象，通过培养基优化，从其发酵产物中分离鉴定新颖结构的化合物，通过体外抗肿瘤筛选获得先导化合物，并对其进行动物模型抗肿瘤初步药效学考察。累计从六株海洋放线菌菌株的发酵产物中分离鉴定了50个化合物，其中新结构化合物32个，新骨架化合物2个。筛选获得能够选择性且显著地抑制白血病细胞系（NB4、BC-3、JSC、DB）的化合物grincamycin B，其IC50值在0.17-0.28 μM之间，能显著抑制人外周血T细胞（Jurkat）的化合物P-1894B和saquayamycin B，其IC50值分别0.01及0.04 μM，能显著抑制人结肠癌细胞HCT-116的化合物A-7884，其IC50值为0.48 μM。利用白血病裸鼠模型，对体外活性显著的化合物grincamycin B进行了初步的药效学评价。本项目的研究成果，为抗白血病药物的开发提供了先导化合物，为开发利用海洋微生物资源提供理论支持和技术储备。.本项目累计发表论文7篇，包括SCI论文6篇、核心期刊论文1篇，均标注本项目基金号；其中项目负责人为第一作者或通讯作者SCI论文4篇，发表在国际权威天然产物期刊Journal of Natural Products（二区）论文2篇；申请专利3项，其中1项获得授权。